Intervertebral discs (IVDs) in the spine consist of an inner nucleus pulposus (NP) and an outer annulus fibrosus (AF). Low back pain is a common problem that is often treated by surgically removing herniated disc tissue, leaving a defect in the AF. In order to return some function to the disc, several defect closure methods have been developed, such as sutures and barriers, which provide mechanical support for the defect but do not promote biological healing. Various tissue engineered approaches are currently being developed that employ biomaterials as scaffolds for AF-mimicking constructs. These advances are promising but their performance in an AF defect has not been reported. Our approach uses both in vitro and in vivo AF defect models to mechanically and biologically evaluate a biomaterial for AF repair. The AF is comprised of primarily type I collagen and collagen hydrogels have been used for many tissue engineering applications. As a gel, collagen is injectable and can be delivered easily to irregular defects; however, it typically exhibits low stiffness. Our lab has used collagen gels in a total disc replacement that exhibited good integration with surrounding AF tissue (Bowles et al., 2011). Therefore we chose type I collagen hydrogels as our platform for AF repair. We will assess the performance of collagen hydrogels for AF repair through three aims encompassing both in vitro and in vivo studies. The first aim will be to evaluate the effects of gel density and crosslinking on the mechanical contribution of collagen gels delivered to AF defects in vitro. It has been shown that the stiffness of collagen hydrogels increases with collagen density (Ibusuki et al., 2007). Likewise, crosslinking with a nontoxic agent like riboflavin increases gel stiffness. We will screen formulations with different collagen gel densities and riboflavin concentrations to find the combination that yields the stiffet gel, then mechanically test that gel in an AF defect. Aims 2 and 3 focus on long term AF repair and assessing collagen gel integration in an in vivo defect model. More specifically, the goal of Aim 2 is to evaluate the ability of unseeded collagen gels taken from Aim 1 outcomes to preserve disc height and NP content. Collagen gels for total disc replacement have shown increased integration when seeded with ovine AF cells (Bowles et al, 2011; Bowles et al., 2010). Therefore Aim 3 will focus on assessing the effect different cell densities on gel integration with surrounding native AF tissue. When these studies are complete, new methods for the assessment of injectable biomaterials will be established and the performance of high-density collagen for AF repair will be characterized.